DE1081605B - Process for the production of a stable, orally applicable poliomyelitis vaccine - Google Patents

Description

Process for the production of a stable, orally applicable poliomyelitis vaccine The invention relates to the production of a stable, orally applicable poliomyelitis vaccine.

Using a variety of well-known media, such as chicken embryo, Rodent brains and spinal cords and tissue cultures are known to have the poliomyelitis virus so adapted that it can develop and has become non-pathogenic. In general is the activity of the virus when frozen in the presence of moisture stable. This stability of the activity decreases with increasing temperature.

For example, at around 40 ° C, the poliomyelitis virus dies in the generally in about 3 days. At normal cooling temperatures (4 ° C) this is Virus activity less stable than when frozen. Although the polio virus is stable in the frozen state, it becomes during the freeze drying process essentially inactivated.

It is already known to produce any dry vaccine by that one of water-insoluble salts of the elements of group II of the periodic table Adsorbed viruses with antibiotics or sulfonamides until they are completely attenuated the infectious components treated and the vaccine thus obtained isolated and dries, if necessary freeze-dried.

You still have live poliomyelitis vaccines orally in the Way applied that one after thawing a poliomyelitis virus suspension from the frozen state in milk. The virus was also called polyethylene glycol suspen. sion filled in capsules for oral use. All of these uses require however, because of the instability of the activity of the virus when the finished ones are left to stand Preparation in milk, polyethylene glycol or other media or in unfrozen Condition of a preparation immediately before administration. There are many other suggestions been made. However, none of them resulted in an oral solution that was active, stable vaccine.

E, s is therefore required, a carrier or a suspending medium to find, which keeps the activity of the virus stable and suitable for capsule filling is.

Preferably it should be dry, free flowing Act as a suspension medium. The problems that arise when filling, distributing, storing and applying a poliomyelitis virus vaccine have shown advances severely hampered immunization against poliomyelitis virus.

It has now been found that a stable, orally applicable and Poliomyelitis vaccine suitable for capsule filling can be obtained if a liquid Suspension of an attenuated, non-pathogenic poliomyelitis virus and ground, non-hydrated gelatin with a particle size corresponding to a clear Mesh size of about 0.15 to 2.00 mm in the weight ratio to gelatin Virus suspension from 10: 1 to 1: 4 mixed together at around -5 to + 20 ° C, thereby hydrating the gelatin particles, absorbing the virus and a Form free flowing, granular vaccines.

Suspensions of any of the previously known poliomyelitis virus species in their culture media can in this way with non-hydrated gelatin can be combined to form a hydrated, free-flowing, capsule-filled, granular poliomyelitis virus vaccines whose virus activity is stable for a very long time remain. Although the vaccine made according to the invention is hydrated, it remains they are essentially dry in appearance, as evidenced by their granular, free-flowing properties Quality is proven.

This vaccine makes the capsule filling of the poliomyelitis virus and the storage of the capsulated vaccine for long periods of time without inconvenience Effects on the stability of the activity possible. One can use such an orally Prepare poliomyelitis vaccines in large quantities, not immediately before Time of administration needs to be established. The virus activity of this Poliomyelitis Vaccine in capsules is available at room temperature, i.e. at room temperature. H. at about 20 ° C, as well as usual cooling temperature, d. H. at about 4 ° C, several Stable for months.

A culture medium is used to produce the vaccines according to the invention contains an attenuated poliomyelitis virus. by agitation with the gelatin mixed. During exercise, the gelatin hydrates with the growth medium, containing the virus so that the virus is absorbed by the gelatin. the Vaccine changes from an initially thin paste as it hydrates to one consisting of discrete, separate, hydrated particles Dimensions. The vaccine so produced is free flowing and granular and contains the Virus essentially evenly distributed.

In general, the titer of the virus will be between 103 and 108. The titer of the preferred oral dose of virus is between about 104 and 10 '. After culturing in the tissue culture medium of the following examples (virus medium No. 1) poliomyelitis virus generally has titers of about 106 to 109.

The preferred titer for oral administration is obtained by dilution. After culturing in rodent brain or spinal cord medium according to the following In the examples (virus medium No. 2), the poliomyelitis virus generally has titer between approximately 103 and t06 For the process according to the invention, gelatin can be used with a particle size of approximately 0.15 to 2.0 mm clear mesh size, preferably from 0.19 to 0.250 mm clear mesh size, can be used.

One can find numerous antibacterial and antifungal agents add to the virus-containing medium before processing with the gelatin. So became Found useful in the present invention, glycerin, one of the several hexavalent alcohols such as Sortit, Dulcit or Mannitol, Nystatin, (antibiotic from Streptomyces noursei), p-naphthol.

Mixtures of the methyl and propyl esters of p-oxybenzoic acid, for example a mixture of 1 to 5 parts by weight of the methyl ester with 1 part by weight of the Propylesters, alone or mixed together with the virus and its Use breeding media. For example, it's cheap.

Nystatin or equivalent to that used to grow the virus Add medium before incubation. After incubation but before processing with gelatin, glycerin or an equivalent is added. Example of Antibacterial agents used are penicillin, tetracycline, chlortetracycline or streptomycin. These antibacterial and fungicidal agents are used together with the water, the virus and the other substances in the virus-containing media are contained, absorbed during hydration of the gelatin. The thus obtained, Free flowing, granular vaccine can be made using conventional methods common devices in either soft-walled or hard-walled gelatin capsules be bottled.

To further increase the free flowability of the invention Manufactured vaccines can use various known aids before the capsule filling be added. For example, you can starch, such as potato, Milo or corn starch, according to the invention to facilitate the filling of capsules into soft-walled capsules use.

The amounts of gelatin and virus suspension used can be considerable vary. For example, you can use the vaccine using 10 parts by weight Gelatine to one volume of the virus suspension (25 to 75 percent by volume glycerin, preferably about 50 / o). But you can also use one part by weight of gelatin on 4 parts by volume Use virus suspension. If you increase the amount of gelatin considerably beyond the 10: 1 ratio described above, no uniform distribution is obtained of the virus in the gelatin particles as there is not enough fluid to begin with complete wetting of the entire surface of the gelatin particles is present. On the other hand, if the amount of gelatin is reduced below the 1: 4 ratio, then becomes the composition is lumpy and moist and can only be handled with difficulty will. The preferred range for all three poliomyelitis virus types is between 1 and 2 parts by weight of gelatin to 3 parts by volume of virus suspension. If an aid to be added to the composition, it may be in an amount of approximately 5 to 20 percent by weight, calculated on the total weight of gelatin and virus suspension, take place. An amount of about 10 to 12 percent by weight is preferred.

In the following are some essential points of the invention Procedure reproduced. To facilitate filling of the capsule, the temperature should all ingredients within a fairly narrow range of about -5 to about + 20 ° C. For best results, there is a working temperature of about 3 to 5 ° C is required. In addition, the gelatin should preferably be used be added in small portions with agitation to the virus-containing medium. It it is also possible to add the medium to the gelatin. However, if you go into this Way ahead, one must be careful. that adding slowly with vigorous agitation is going on to avoid the extremely rapid hydration that one manufacture of large gelatin aggregates that are incompletely hydrated.

If the temperature limits given above are not observed, this gives an unsatisfactory product. If you cross the upper one Temperature limit (20 ° C) so will be large, sticky, incompletely hydrated Gelatin particles obtained as a result of too rapid hydration. With these higher ones Temperatures, the large particles that are unsuitable for filling capsules, In terms of weight, they exceed the finer particles suitable for filling capsules. You can only be sure when using temperatures of 10 ° C and below that one obtains completely satisfactory results. If you use temperatures, which are below -5 ° C, virus-containing solid masses separate from the liquid Phase off. It is therefore preferable to work at above 0 ° C. Gelatin with a particle size outside a range of about 0.15 to 2.0mm Mesh size is unsuitable for capsule filling after hydration.

The following examples illustrate the invention. It is clear that equivalent procedural measures and amounts can be used, for example chicken embryo can be used as a growth medium for the non-pathogenic, attenuated poliomyelitis virus use.

Example 1 1. The following is a description of one typical procedure for preparing a tissue culture medium and its use for the cultivation of poliomyelitis virus type I (SM strain), type II (TN strain) or type III (Fox strain) and 2. a typical manufacture of a mouse brain and Spinal cord medium and its use in the manufacture of attenuated poliomyelitis virus of the species and strains described under 1.

In the following examples, these media are identified as virus-containing Medium No. 1 and virus-containing medium No. 2 are designated.

(1) Virus-containing medium No. 1 A. Growth of monkey kidney cells (cf. Virology, Vol. 2 [August 1956], pp. 575 and 576) Kidneys removed from rhesus or cynomolgus monkeys are chopped into small pieces and kept overnight (approx Hours) digested at 4 ° C with 0.25% trypsin solution and obtained living cells from it. Approximately 150,000 of these cells per ccm are suspended in a tissue culture with the following composition: Bovine serum ultrafiltrate ... 15 parts by volume of Hank's balanced salt solution 80 parts by volume of bovine embryo extract ... 5 parts by volume of penicillin ...... ........... 50 units / ml liquid streptomycin .............. 50 micrograms / ml liquid Hank balanced salt solution: Na 8.0 g K 0.4 g CaCl2 (anhydrous) ........ 0.2 g Mgso4. 7H20 .......... 0.2 g per 1000 ml Na2 HP 04 2 H2 0 0.06 g water KH2PO4 ................... 0.06 g Glucose 1.00 g Nua 35 go The cell suspension obtained is filled into 1-1 Povitsky bottles and incubated at 370.degree. After 6 days, the cells adhere to the glass container. They form a complete layer of cells at the bottom of the container.

B. Growth of Virus After 6 days, the liquid contents of the tissue culture medium become removed from the bottles and replaced with 500ml of # 199 tissue culture medium (See "Reagents & Media for Tissue Culture & Virus Propagation", published from Difco). The contents of the bottles are then each with 5 ml attenuated poliomyelitis virus inoculated (see Journal of the American Medical Association, Vol. 162, [December 1, 1956], P. 1281). The inoculated medium and the cells are incubated at 37 ° C. If in essentially all cells are destroyed, d. H. when they no longer stick to the glass, the fluid medium and cell debris are harvested. This will in general made at the end of the fourth day. After harvesting, the harvested virus-containing Medium No. 1 was tested for its potency by titration in tissue culture tubes. It can then be mixed with gelatin to make a free flowing, granular vaccine are processed. However, if desired, it can also be frozen and up to desired processing can be stored with the gelatin.

(2) Virus-containing medium No. 2 Swiss albino mice are each 0.03 ml of an attenuated virus was entered intracerebrally via the spinal cord. if the mice are paralyzed, the brains and spinal cords of the animals are removed. The removed brains and spinal marks are then made into a suspension (Weight/ Volume) made in normal saline, to which 50 micrograms of streptomycin and 50 units of penicillin / ml of liquid are added. This medium represents the virus-containing medium no. 2, which, like the virus-containing medium no. 1 processed with gelatin or frozen and stored until desired use can be.

Example 2 A Poliomyelitis Virus Vaccine Type I (SM strain) was made from the following ingredients: Gelatin (0.250-0.177mm clear mesh size) ................ 50 g corn starch 13 g virus-containing medium no. 1 ........ 37.5 ccm glycerine ................... 37.5 ccm virus-containing medium no. 1 and glycerine were combined to form the virus suspension. Before mixing became suspension and gelatin separately cooled in ethanol-dry ice baths to approximately 4 ° C. The gelatin was then agitated at approximately 4 ° C to produce individual hydrated particles soaked with the virus suspension.

The corn starch was then mixed into the composition mixed in.

The so prepared, hydrated, free flowing, granular vaccine was then filled into hard-walled gelatine capsules and their effectiveness was determined.

The results are as follows: Titer of vaccine activity Temperature time in days 0 1 1 1 7 1 13 + 27 ° C 104, s 104, 5 + 4 ° C 104.3 104.5 104.5 1042.2 -15 ° C 104.3 105, 2 Example 3 A poliomyelitis virus vaccine of type I (SM strain) was prepared according to the procedure of Example 2 from the following amounts of material: Gelatin (0.250 to 0.177 mm mesh size) ........ ........ 23, 8 g corn starch 6, 8 g virus suspension No. 2 (50%) and glycerin (50%) 34 ccm All ingredients and additives with the exception of the corn starch were dissolved before mixing Chilled 4 ° C. The gelatin was slowly hydrated as in Example 2 with agitation at low temperatures.

When completely hydrated, the corn starch was made in three portions inflicted during movement. As in Example 2, a free-flowing, granular one was obtained Vaccine with an effective titer.

Example 4 A Poliomyelitis Virus Vaccine Type II (TN Strain) of effective titer and free-flowing, granular form was according to Example 2 using the following ingredients: Gelatin (0.250 to 0.177 mm clear mesh size) ................ 14.05 g corn starch 14.05 g virus suspension No. 2 (50 "/ o) and glycerine (50%) 56.3 cc The hydration temperature was 4 ° C. After complete hydration, the corn starch was divided into three portions admitted while moving.

Example 5 A Type II Poliomyelitis Vaccine (TN Strain) of Effective The titer and free flowing, granular form were obtained from Example 2 from the following Components prepared: Gelatine (0.250 to 0.149mm clear mesh size) .................. 70 g corn starch 20 g virus-containing medium No. 1 ........ 50 ccm glycerine ........................... 50 cc. After mixing the liquid ingredients at 4 ° C, the corn starch became admixed until individual particles were formed.

Example 6 A type III poliomyelitis virus vaccine was used (Fox strain) of effective titer according to and using the procedure Components and amounts with the exception of the virus itself, d. H. of gelatin, starch, Glycerin and medium no. 1 as prepared in Example 2. A hydrated, free-flowing, granular vaccines, which after filling in soft-walled capsules with of tissue culture titration had an effectiveness similar to that of the example 2 was.

Example 7 A Type III Poliomyelitis Virus Vaccine (Fox Strain) of effective titer was determined according to the Procedure and using the same Components and amounts with the exception of the virus itself, d. H. of gelatine, glycerine, Starch and medium no. 2 as prepared in example 3. A hydrated, free-flowing, granular vaccine that has been filled into hard-walled capsules. at the tissue culture titration showed this vaccine an activity similar to that of Example 3.

Claims (3)

PATENT CLAIMS: 1. Method of producing a stable, oral applicable poliomyelitis vaccine, which is suitable for capsule filling characterized in that one is a liquid suspension of an attenuated, non-pathogenic Poliomyelitis Virus and ground, unhydrated gelatin of a particle size corresponding to a clear mesh size of approximately 0.15 to 2.00 mm in weight ratio Gelatin to virus suspension from 10: 1 to 1: 4 at about -5 to + 20 ° C with each other mixes. 2. The method according to claim 1, characterized in that the components at 0 to 10 ° C, preferably at 3 to 5 ° C, and preferably with shaking together be mixed. 3. The method according to claim 1 or 2, characterized in that the gelatin used has a particle size corresponding to a clear mesh size from 0.19 to 0.250 mm and in small portions with shaking to the liquid virus suspension is added. Publications considered: German Patent No. 956 975.